Method and composition for hypothermic storage of placental tissue

ABSTRACT

A tissue storage solution includes hypothermic storage compositions and methods. The hypothermic storage composition includes media containing Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) and albumin. A method of hypothermically storing tissue includes storing such tissue in a storage medium including DMEM and albumin. A method for wound or defect treatment includes applying tissue, stored in a hypothermic storage medium containing DMEM and albumin, to the site of such wound or defect.

FIELD OF THE INVENTION

The present invention is directed to hypothermic storage compositionsand methods. More particularly, the present invention is directed to ahypothermic storage solution including Dulbecco's modified Eagle'smedium (DMEM) and human plasma albumin, a method of hypothermicallystoring tissue, such as placental membrane, in the solution, and amethod for wound treatment by applying tissue stored in the solution tothe site of a wound.

BACKGROUND OF THE INVENTION

The placenta surrounds a fetus during gestation and is composed of,among other tissues, an inner amniotic layer that faces the fetus and agenerally inelastic outer shell, or chorion. The placenta anchors thefetus to the uterine wall, allowing nutrient uptake, waste elimination,and gas exchange to occur via the mother's blood supply. Additionally,the placenta protects the fetus from an immune response from the mother.From the placenta, an intact placental membrane comprising the amnionand chorion layers can be separated from the other tissues. The amnionis the innermost layer of the placenta and consists of a thick basementmembrane and an avascular stromal matrix.

Clinicians have used intact placental membrane, comprising an amnion anda chorion layer, in medical procedures since as early as 1910 [Davis J.S., John Hopkins Med J, 15:307 (1910)]. The amniotic membrane, whenseparated from the intact placental membrane, may also be used for itsbeneficial clinical properties [Niknejad H., et al. Eur Cell Mater,15:88-99 (2008)]. Certain characteristics of the placental membrane makeit attractive for use by the medical community. These characteristicsinclude, but are not limited to: its anti-adhesive, anti-microbial, andanti-inflammatory properties; wound protection; its ability to induceepithelialization; and pain reduction [Mermet I., et al. Wound RepRegen, 15:459 (2007)].

Other uses for placental membrane include its use for scaffolding orproviding structure for the regrowth of cells and tissue. An importantadvantage of placental membrane in scaffolding is that the amnioncontains an epithelial layer. The epithelial cells derived from thislayer are similar to stem cells, allowing the cells to differentiateinto cells of the type that surrounds them. Multipotent cells similar tostem cells are also contained within the body of the amniotic membrane.Additionally, the amniotic membrane contains various growth and trophicfactors, such as epidermal, insulin-like, and fibroblast growth factors,as well as high concentrations of hyaluronic acid, which may bebeneficial to prevent scarring and inflammation and to support healing.Thus, placental membrane offers a wide variety of beneficial medicaluses.

Cell-based therapies have considerable potential for the repair andregeneration of tissues. The addition of a scaffold to these cell-basedtherapies has yielded improved outcomes [Krishnamurithy G., et al. JBiomed Mater Res Part A, 99A:500-506 (2011)]. Ideally, the material usedfor the scaffold will be biocompatible such that it provokes little tono immune response, biodegrades, and is available in sufficientquantities to be practical. Although the placental membrane has longbeen identified as a material potentially filling this role in theclinic, efforts have been limited to in vitro studies, impractical invivo techniques, or have yielded less-than-optimal outcomes.Furthermore, the conditions under which the scaffold is used may have adramatic effect on the therapeutic efficacy.

A number of placental membrane products have been studied in theliterature and used clinically, falling into two primary categories. Thefirst category involves the use of the intact membrane, be it fresh,dried, freeze-dried, cryopreserved, or preserved in glycerol or alcohol.In this formulation, the membrane is useful for a number of purposes,but is not suitable for others, such as applications requiringinjection, or the filling of a space which does not conform to the thinplanar shape of the membrane itself.

The second category involves the grinding, pulverizing and/orhomogenizing of the membrane into small particles, which may then beresuspended in solution. Such techniques are described, for example, inU.S. patent application Ser. Nos. 11/528,902; 11/528,980; 11/529,658;and 11/535,924. This grinding may be done dry or wet, and temperatureduring grinding may or may not be controlled, such as in the case ofcryogrinding. Products produced using this method are useful for anumber of applications, and may be injected under appropriateconditions. However, they have several deficiencies for certainapplications. First, the cells contained in the placental membranes willbe destroyed during the grinding process. Second, proteins and growthfactors in the membrane may be leached out or lost during this process,including any subsequent washing or other treatment of the groundparticles. Indeed, the removal of potentially angiogenic factors such asgrowth factors may be an objective of this type of processing. Third,resuspension of these small particles in typical physiologic solutions,such as saline, results in a free-flowing fluid with low viscosity. Uponinjection or placement, this fluid may dissipate rather than remain inthe desired treatment location. Fourth, the resulting fragments may notbe large enough to permit cell engraftment and proliferation, if sodesired.

However, amniotic membrane preparations have been shown to havesignificant beneficial bioactivity. Many of the cells contained in thesemembranes are multi- or pluri-potent. The membranes also contain a richsource of growth factors, as well as hyaluronic acid, collagen, andother factors which have been shown to support tissue healing. Amnioticmembrane has been shown to attract and to stimulate the proliferation ofcells involved in tissue healing, such as mesenchymal stem cells (MSCs)and fibroblasts.

Articular cartilage, located on the articular ends of bones at jointsthroughout the body, is composed of hyaline cartilage and containsrelatively few chondrocytes that are embedded in extracellular matrixmaterials, such as type II collagen and proteoglycans [Moriya T., et al.J Orthop Sci, 12:265-273 (2007)]. Articular cartilage has a limitedability to self-repair, in part due to the avascular characteristics ofthe cartilage, which poses a significant challenge to treating jointinjuries and diseases. The repair of cartilage defects in humans cantherefore be a difficult endeavor, and multiple options exist for thesurgeon to treat such defects. The surgeon may choose to influence thedefect with microfracture, abrasion or other marrow stimulationtechniques which stimulate bleeding of the subchondral bone and thegeneration of a clot and ultimately a fibrocartilage patch which fillsthe defect. Other options allow for the filling of the defect withchondrocytes of variable sources, both of autograft and allograftorigin.

A key advantage of marrow stimulation techniques over most otheravailable therapies is that marrow stimulation may be carried outarthroscopically using a relatively simple surgical technique, withminimal disruption to the joint and surrounding tissues [Mithoefer K.,et al. J Bone Joint Surg Am, 88(Suppl 1 Pt 2):294-304 (2006)]. Thetechnique is also cost-effective. Efforts have therefore been made toimprove the outcome of marrow stimulation techniques. Ground cartilage,either autograft or allograft (e.g. the product commercially marketed asBioCartilage), has been proposed for this purpose [Xing L., et al. KneeSurg Sports Traumatol Arthrosc, 21:1770-1776 (2013)]. However the use ofautograft cartilage requires additional operative steps and donor sitemorbidity. Further, the use of allograft cartilage alone has not yieldedsatisfactory results.

Current treatments, including cell-based therapies, have resulted in thegeneration of undesirable fibrocartilaginous tissue rather than hyalinecartilage [Diaz-Prado S. M., et al. BIOMEDICAL ENGINEERING, TRENDS,RESEARCH, AND TECHNOLOGIES, pp. 193-216 (2011)]. As such, there remainsa significant clinical need for therapies capable of repairing damagedarticular cartilage by specifically regenerating hyaline-like cartilage.

A similar need exists for solutions for the repair of meniscal defects.A meniscus is a crescent-shaped fibrocartilaginous structure that, incontrast to articular discs, only partly divides a joint cavity. Inhumans they are present in the knee, acromioclavicular,sternoclavicular, and temporomandibular joints. Generally, the term‘meniscus’ refers to the cartilage of the knee, either to the lateral ormedial menisci. Both are cartilaginous tissues that provide structuralintegrity to the knee as it undergoes tension and torsion. They areconcave on the top and flat on the bottom, articulating with the tibia.They are attached to the small depressions (fossae) between the condylesof the tibia (intercondyloid fossa), and towards the center they areunattached and their shape narrows to a thin shelf. The blood flow ofthe meniscus is from the periphery to the central meniscus. Blood flowdecreases with age and the central meniscus is avascular by adulthoodleading to very poor healing rates. Meniscal defects are repaired usingsutures or other fixation approaches. Partial meniscectomies are alsocommonly used [Kon E., et al. Tissue Eng Part A, 18(15-16): 1573-1582(2012); Fiorentino G., et al. Arthrosc Tech, 2(4):e355-e359 (2013);Scotti C., et al. Eur Cell Mater, 26:150-170 (2013)].

Another related problem involves the regeneration of the humanintervertebral disc. Intervertebral discs are fibrocartilaginous tissuesoccupying the space between vertebral bodies in the spine. They transmitforces from one vertebra to the next, while allowing spinal mobility.The structural properties of the disc are largely dependent on itsability to attract and to retain water. Proteoglycans in the disc exertan osmotic “swelling pressure” that resists compressive loads.Degeneration of the intervertebral disc is a physiologic process that ischaracteristic of aging in humans. With age, the disc undergoes avariety of changes, the most notable being a loss of proteoglycancontent resulting in reduced osmotic pressure and a reduction in discheight and ability to transmit loads [Park S. H., et al., Tissue EngPart A, 18(5-6):447-458 (2012)]. Disc degeneration is an important anddirect cause of spinal conditions that account for most neck and backpain. As is the case with the related cartilage cells, components of theamniotic membrane may promote healing and recovery of the intervertebraldisc and associated cells.

The storage and transport of placental membranes are subject to numerousregulatory schemes. For example, the membranes must be stored for aspecific period of time before use in a subject (i.e., fourteen daysbefore use), during which time it is periodically tested forcontamination from bacteria, viruses and other non-placental cell types.Additional delay is caused by the transport of the membrane to theappropriate medical facility. This delay may reduce viability of theplacental membrane cells. To maintain cell viability within the amnioticmembrane, it must be stored and transported in cell culture mediacontaining appropriate supplements. These supplements should be selectedto maximize cell viability and to extend the time in which the tissuecan be reliably stored. The membrane storage media should meet currentlyexisting clinical standards to guarantee the safety of the products andensure regulatory compliance. The media therefore must be chemicallydefined/cGMP compliant and must be free of both xeno-derived andhuman-derived components.

Traditional tissue storage generally involves cryogenic preservationmethods by which cells or whole tissues are preserved by cooling tosub-zero temperatures. The low temperatures effectively stop anyenzymatic or chemical activity that might cause tissue damage.Cryopreservation methods seek to reach low temperatures without causingadditional damage by the formation of ice during freezing. Traditionalcryopreservation techniques rely on coating the cells or tissues with acryoprotectant to prevent intracellular ice formation. Macroscopic iceformation may still occur, however, causing perforation which affectsthe integrity of the tissue.

Hypothermic tissue storage generally occurs at temperatures abovefreezing, for instance from between 0° C. and 20° C., which prevents icecrystal formation. Many issues relating to cell function and viabilityare associated with hypothermic tissue storage, such as osmolality,ischemia, hypoxia, and oxygen-derived free radicals, as described below.

Osmolality.

Under equilibrium, a higher concentration of inorganic ions are presentin the intracellular space as opposed to the extracellular space, aphenomenon known as the Donna Effect [Dick, D. A. T., Relation betweenwater and solutes: Theory of osmotic pressure, in CELL WATER, E. E.Bittar, Editor 1966, Butterworths: London. pp. 15-43.]. Therefore, atequilibrium, there is a tendency for cells to uptake water. Cells managetheir osmolality via active ion transport in which ions, such as sodiumand chloride, are transported out of the membrane via pumps to preventwater intake and subsequent lysis. Osmolality is a colligative propertyand dependent on the amount of ions present within solution as opposedto their size or nature [Taylor M. J., Physico-chemical principles inlow temperature biology, in THE EFFECTS OF LOW TEMPERATURES ONBIOLOGICAL SYSTEMS, B. W. W. Grout & G. J. Morris, Editors. 1987, EdwardArnold: London. pp. 3-71.]. The ATP consumption required to perform theactive transport reactions are adapted to occur at normal bodytemperature. At hypothermic temperatures, the active transport pumps areunable to bind to ATP to process it as energy. In addition, underhypothermic conditions, high energy reserves are depleted whenmitochondrial energy transduction (production of ATP) fails. Theactivity of the sodium and potassium pumps at 5° C. is approximately 1%of its normal levels at physiological temperatures [Ellory J. C. &Willis J. S., Phasing out the sodium pump, in EFFECTS OF LOWTEMPERATURES ON BIOLOGICAL MEMBRANES, G. J. Morris and A. Clarke,Editors. 1981, Academic Press: London. pp. 107-120.]. Normal osmolalityof the extracellular fluid is approximately 280 to 310 mOsmol/L. Becausethe sodium and potassium pumps fail under hypothermic conditions,hypothermic storage media should be slightly hypertonic to counteractthis change.

Ischemia/Hypoxia.

An immediate consequence of cessation of blood supply to an organ is theloss of oxygen supply to the tissue. Oxygen is necessary for the processof aerobic respiration and the production of adenosine triphosphate(ATP), the main energy source utilized by cells. In the absence ofoxygen, ATP is depleted within a few minutes. This depletion leads to ashift from aerobic to anaerobic metabolism which is self-limiting andcauses the production of lactate and protons. This leads to a cascadeeventually culminating in necrosis and cell death.

For every 10° C. drop in temperature, cellular oxygen consumptiondeclines 50% [Taylor M. J., Biology of Cell Survival in the Cold: TheBasis for Biopreservation of Tissues and Organs, in ADVANCES INBIOPRESERVATION, J. G. Baust & J. M. Baust, Editors. 2007, CRC Press:Boca Raton, Fla.]. This produces slower reaction rates and a decrease incellular metabolism.

Oxygen-Derived Free Radicals (ODFRs).

The cooling of cells increases their susceptibility to produce freeradicals while attenuating the mechanisms by which cells normally dealwith free radical formation [Fuller B. J., Gower J. D., & Green C. J.,Cryobiology, 25(5):377-393 (1988)]. Low concentrations of molecularoxygen, such as those found in tissue preservation solutions, aresufficient for the development of ODFRs. For this reason, naturalpharmacological scavengers such as SOD, catalase, or mannitol mayimprove the viability of hypothermically stored organs [Fuller, et al.Cryobiology, 23(4):358-365 (1986)].

SUMMARY OF THE INVENTION

The present invention is directed to both compositions and methodsrelated to the hypothermic storage of tissue. According to one aspect ofthe invention, there is provided a composition of hypothermic storagemedia that includes DMEM and albumin. In another aspect of theinvention, there is provided a method of hypothermically storing tissue.The method includes the steps of preparing or obtaining media containingDMEM and albumin, placing tissue into the media, and storing the tissueand media hypothermically. In yet another aspect of the invention thereis provided a method for wound treatment. The method includes the stepsof storing tissue in hypothermic storage media and applying the tissueto the site of the wound. The tissue may also be minced or ground intoparticles, which may be injected into the site of the wound.

A further understanding of the nature and advantages of the presentinvention will be realized by reference to the remaining portions of thespecification and the drawings of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure can be better understood with reference to the followingdrawings. The elements of the drawings are not necessarily to scalerelative to each other, emphasis instead being placed upon clearlyillustrating the principles of the disclosure. Furthermore, likereference numerals designate corresponding parts throughout the severalviews.

FIG. 1A is a graph illustrating preliminary cell viability assessment.

FIG. 1B is a quantitative image of cell viability.

FIG. 1C is an additional quantitative image of cell viability.

FIG. 1D is an additional quantitative image of cell viability.

FIG. 2A is a graph showing the viability of placental membrane cells at42 days.

FIG. 2B is an additional graph showing the viability of placentalmembrane cells at 42 days.

FIG. 2C is an additional graph showing the viability of placentalmembrane cells at 42 days.

FIG. 3 is an illustration of qualitative assessment of cell viabilityover time.

FIG. 4A is an illustration of the macroscopic handling characteristicsof tissue stored in a DMEM solution.

FIG. 4B is an illustration of the macroscopic handling characteristicsof tissue stored in a DMEM+recombinant human albumin (rhA) solution.

FIG. 5A is an illustration of histological staining of tissues stored ina DMEM solution for four weeks.

FIG. 5B is an illustration of histological staining of tissues stored ina DMEM+rhA solution for four weeks.

FIG. 5C is an illustration of histological staining of tissues stored ina DMEM solution for six weeks.

FIG. 5D is an illustration of histological staining of tissues stored ina DMEM+rhA solution for six weeks.

FIG. 5E is a graph illustrating the total protein content of placentalmembrane cells.

FIG. 5F is a graph illustrating the concentration of tissue inhibitingmatrix metalloproteinases in placental membrane cells.

FIG. 6A is a graph illustrating stress testing on cells incubated in thestorage solution for nine weeks.

FIG. 6B is a graph illustrating strain testing on cells incubated in thestorage solution for nine weeks.

FIG. 6C is a graph illustrating modulus testing on cells incubated inthe storage solution for nine weeks.

FIG. 7A is a guide illustrating the placement of the experimentalplacental membranes over wounds.

FIG. 7B is an illustration of wounds treated with experimental placentalmembranes nine days post-procedure.

FIG. 7C is an additional illustration of wounds treated withexperimental placental membranes nine days post-procedure.

FIG. 7D is an additional illustration of wounds treated withexperimental placental membranes nine days post-procedure.

FIG. 8 is a chart listing the results of the histological examination ofwounds treated with experimental placental membranes nine dayspost-procedure.

FIG. 9A is an illustration of wounds treated with experimental placentalmembranes twenty-one days post-procedure.

FIG. 9B is an additional illustration of wounds treated withexperimental placental membranes twenty-one days post-procedure.

FIG. 9C is an additional illustration of wounds treated withexperimental placental membranes twenty-one days post-procedure.

FIG. 9D is an additional illustration of wounds treated withexperimental placental membranes twenty-one days post-procedure.

FIG. 10 is a chart listing the results of the histological examinationof wounds treated with experimental placental membranes twenty-one dayspost-procedure.

FIG. 11A is an illustration of the histological examination of a woundtreated with dehydrated amnion twenty-one days post-procedure.

FIG. 11B is an illustration of the histological examination of a woundtreated with fresh hypothermically stored placental membrane twenty-onedays post-procedure.

FIG. 11C is an illustration of the histological examination of anuntreated wound twenty-one days post-procedure.

FIG. 11D is an illustration of the histological examination of a woundtreated with dehydrated mesh twenty-one days post-procedure.

FIG. 12A is an additional illustration of the histological examinationof a wound treated with dehydrated mesh twenty-one days post-procedure.

FIG. 12B is an additional illustration of the histological examinationof a wound treated with dehydrated mesh twenty-one days post-procedure.

FIG. 12C is an additional illustration of the histological examinationof a wound treated with dehydrated amnion twenty-one dayspost-procedure.

FIG. 12D is an additional illustration of the histological examinationof a wound treated with dehydrated amnion twenty-one dayspost-procedure.

FIG. 13A is a quantification of abnormal tissue in wounds treated withexperimental placental membranes at nine and twenty-one dayspost-procedure.

FIG. 13B is a quantification of basket-weave matrix in wounds treatedwith experimental placental membranes at nine and twenty-one dayspost-procedure.

FIG. 13C is a quantification of follicle/gland formation in woundstreated with experimental placental membranes at nine and twenty-onedays post-procedure.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions, articles, devices, and/or methods aredisclosed and described, it is to be understood that they are notlimited to specific methods unless otherwise specified, or to particularreagents unless otherwise specified, and as such may vary. It is also tobe understood that the terminology as used herein is used only for thepurpose of describing particular embodiments and is not intended to belimiting.

This application references various publications. The disclosures ofthese publications, in their entireties, are hereby incorporated byreference into this application to describe more fully the state of theart to which this application pertains. The references disclosed arealso individually and specifically incorporated herein by reference formaterial contained within them that is discussed in the sentence inwhich the reference is relied on.

A. DEFINITIONS

In this specification, and in the claims that follow, reference is madeto a number of terms that shall be defined to have the followingmeanings.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “the wound” includes two or more such wounds, andthe like.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. When such a range isexpressed, an embodiment includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by the use of “about,” it will be understood that theparticular value forms another embodiment. It will be understood thatthe endpoints of each of the ranges are significant both in relation tothe other endpoint and independently of the other endpoint. It will alsobe also understood that there are a number of values disclosed herein,and that each value is also disclosed herein as “about” that particularvalue in addition to the value itself. For example, if the value “50” isdisclosed, then “about 50” is also disclosed. It is also understood thatwhen a value is disclosed that “less than or equal to” a value, thatvalues “greater than or equal to the value” and possible ranges betweenvalues are also disclosed, as understood by one skilled in the art. Forexample, if the value “50” is disclosed, then “less than or equal to 50”and “greater than or equal to 50” are also disclosed. It is alsounderstood that the throughout the application, data are provided indifferent formats, and it is understood that these data representendpoints and starting points as well as ranges for any combination ofthe data points. For example, if a particular data point “50” and aparticular data point “100” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 50 and 100 are considered disclosed as well as between 50 and100.

As used herein, “amniotic tissue” means amniotic fluid cells, placentalmembrane, amnion tissue or combinations thereof.

As used herein, the terms “optional” and “optionally” mean that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not occur.

As used herein, the phrase “substantially all” refers to the maximumamount reasonably attainable by one skilled in the art.

As used herein, the phrases “placental membrane” and “amnion tissue”refer to one or more layers of the placental membrane. For example,placental membrane or amnion tissue may refer to a placental membranecomprising both the amniotic and chorionic layers. In another example,placental membrane or amnion tissue may refer to a placental membrane inwhich the chorion has been removed. In another example, placentalmembrane or amnion tissue may refer to a placental membrane in which theepithelial layer has been removed.

As used herein, the terms “treatment” and “treating” include anydesirable effect on the symptoms or pathology of a disease or condition,and may include even minimal reductions in one or more measurablemarkers of the disease or condition being treated. “Treatment” does notnecessarily indicate complete eradication or cure of the disease orcondition, or associated symptoms thereof. The subject receiving thistreatment is any animal in need, including primates, in particularhumans, and other mammals including, but not limited to, equines,cattle, swine, sheep, poultry and pets in general.

B. MAKING OF THE PLACENTAL MEMBRANE PREPARATION

The placental membranes that utilize the present hypothermic storagesolution may be prepared as described previously in U.S. patentapplication Ser. No. 14/212,010, entitled “Preparations Derived FromPlacental Materials and Methods of Making and Using the Same,” U.S.patent application Ser. No. 13/754,742, entitled “Placental MembranePreparation and Methods of Making and Using the Same,” and U.S. patentapplication Ser. No. 13/754,716, entitled “Placental MembranePreparation and Methods of Making and Using the Same,” the contents ofwhich are incorporated herein by reference. Briefly, the placentalmembrane preparation includes amnion tissue and, optionally, amnioticfluid cells. The amnion tissue component of the placental membranepreparation is produced from placentas collected from consenting donorsin accordance with the Current Good Tissue Practice guidelinespromulgated by the U.S. Food and Drug Administration. In particular,soon after the birth of a human infant via a Cesarean section delivery,the intact placenta is retrieved, and the placental membrane isdissected from the placenta. Afterwards, the placental membrane iscleaned of residual blood, placed in a bath of sterile solution, storedon ice and shipped for processing. Once received by the processor, theplacental membrane is rinsed to remove any remaining blood clots, and ifdesired, rinsed further in an antibiotic rinse [Diaz-Prado S. M., et al.Cell Tissue Bank, 11:183-195 (2010)].

The antibiotic rinse may include, but is not limited to, theantibiotics: amikacin, aminoglycosides, amoxicillin, ampicillin,ansamycins, arsphenamine, azithromycin, azlocillin, aztreonam,bacitracin, capreomycin, carbacephem, carbapenems, carbenicillin,cefaclor, cefadroxil, cefalexin, cefalotin, cefamandole, cefazolin,cefdinir, cefditoren, cefepime, cefixime, cefoperazone, cefotaxime,cefoxitin, cefpodoxime, cefprozil, ceftaroline fosamil, ceftazidime,ceftibuten, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime,chloramphenicol, ciprofloxacin, clarithromycin, clindamycin,clofazimine, cloxacillin, colistin, cycloserine, dapsone, daptomycin,demeclocycline, dicloxacillin, dirithromycin, doripenem, doxycycline,enoxacin, ertapenem, erythromycin, ethambutol, ethionamide,flucloxacillin, fosfomycin, furazolidone, fusidic acid, gatifloxacin,geldanamycin, gentamicin, glycopeptides, grepafloxacin, herbimycin,imipenem or cilastatin, isoniazid, kanamycin, levofloxacin, lincomycin,lincosamides, linezolid, lipopeptide, lomefloxacin, loracarbef,macrolides, mafenide, meropenem, methicillin, metronidazole,mezlocillin, minocycline, monobactams, moxifloxacin, mupirocin,nafcillin, nalidixic acid, neomycin, netilmicin, nitrofurans,nitrofurantoin, norfloxacin, ofloxacin, oxacillin, oxytetracycline,paromomycin, penicillin G, penicillin V, piperacillin, platensimycin,polymyxin B, pyrazinamide, quinolones, quinupristin/dalfopristin,rifabutin, rifampicin or rifampin, rifapentine, rifaximin,roxithromycin, silver sulfadiazine, sparfloxacin, spectinomycin,spiramycin, streptomycin, sulfacetamide, sulfadiazine, sulfamethizole,sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole,sulfonamidochrysoidine, teicoplanin, telavancin, telithromycin,temafloxacin, temocillin, tetracycline, thiamphenicol, ticarcillin,tigecycline, tinidazole, tobramycin, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX), andtroleandomycin, trovafloxacin, or vancomycin.

The antibiotic rinse may also include, but is not limited to, theantimycotics: abafungin, albaconazole, amorolfin, amphotericin B,anidulafungin, bifonazole, butenafine, butoconazole, caspofungin,clotrimazole, econazole, fenticonazole, fluconazole, isavuconazole,isoconazole, itraconazole, ketoconazole, micafungin, miconazole,naftifine, nystatin, omoconazole, oxiconazole, posaconazole,ravuconazole, sertaconazole, sulconazole, terbinafine, terconazole,tioconazole, voriconazole, or other agents or compounds with one or moreanti-fungal characteristics.

Although placental membranes possess many benefits and applications,availability of the membranes has limited their use. The amount ofplacental membrane generated from a single birth is small. As would beexpected, because the supply of placental membranes is relatively small,the cost of placental membranes limits their use only to procedures thatsurpass a certain price or complexity. U.S. patent application Ser. Nos.13/250,096 and 13/647,525 describe a placental membrane including aplurality of slits for increasing the membranes capacity to expand. Theslits are provided through the membrane and are provided in sufficientnumbers to produce a mesh-like pattern which enables the membrane to bestretched and therefore increase its length and width.

The placental membrane may be processed to remove one or more particularlayers of the membrane. The chorion may be removed from the placentalmembrane by mechanical means well-known to those skilled in the art. Thechorion may be removed, for example, by carefully peeling the chorionfrom the remainder of the placental membrane using blunt dissection [JinC. Z., et al. Tiss Eng, 13:693-702 (2007)]. Removal of the epitheliallayer from the placental membrane may be achieved using several methodswell-known to those skilled in the art. The epithelial layer may bepreserved or, if desired, may be removed by, for example, using trypsinto induce necrosis in the epithelial cells [Diaz-Prado S. M., et al.Cell Tissue Bank, 11:183-195 (2010)]. Removal of the epithelial layermay comprise, for example, treatment with 0.1%trypsin-ethylenediaminetetraacetic acid (EDTA) solution at 37° C. for 15minutes followed by physical removal using a cell scraper [Jin C. Z., etal. Tiss Eng, 13:693-702 (2007)]. Preferably, the placental membraneutilized for the amnion tissue component of the placental membranepreparation is the amniotic membrane including the amniotic epithelialcell layers but excluding the chorion.

The placental membranes may be ground using techniques known in the art,and the resulting particles either re-suspended in the hypothermicstorage medium described herein or dried. Such processing may be carriedout so as to preserve, to the extent possible, the protein content ofthe membrane, including growth factors. Preferably, grinding should beconducted under temperature-controlled conditions, such as in acryomill. Preferably such ground pieces of tissue should have a particlesize of less than 1 mm. Alternatively, the membranes may be minced usingtechniques known in the art, creating, for example, small cubes ofmembrane tissue. Preferably such minced pieces of tissue have particlesizes ranging from 0.1 mm to 5 mm. Minced tissue particles may besquare, rounded, oblong or irregular in shape.

The ground or minced placental membrane includes amnion tissuecontaining organized amniotic extracellular matrix (ECM), amniotictissue cells and growth factors contained within the ECM and amniotictissue cells. The ECM includes amnion-derived collagen, fibronectin,laminin, proteoglycans and glycosaminoglycans. The amnion-derivedcollagen may be derived from an epithelium layer, a basement membranelayer, a compact layer, a fibroblast layer, an intermediate layer and aspongy layer of the amnion tissue.

The placental membrane preparation may be combined with prenatal stemcells if desired. For example the preparation may include amniotic fluidcells that are derived from amniotic fluid that is collected duringamniocentesis or scheduled C-section from consenting donors. Theamniotic fluid is spun thereby pelletizing the amniotic fluid cells. Theresulting amniotic fluid cells may be combined with ground placentalmembrane and cryopreserved in a solution containing approximately 5 to10% vol/vol Dimethyl Sulfoxide (DMSO) and 15 to 25% vol/vol protein,with the balance being crystalloids.

Minced, ground or morselized membrane particles may be freeze dried andsterilized, or stored in a cryopreservative or hypothermic storagesolution, as described herein, allowing the preservation of theviability of some membrane cells. A suitable ground placental membranepreparation, which includes amniotic fluid cells, is sold by NuTechMedical, Inc. of Birmingham, Ala. under the name NuCel™.

The placental membrane preparation may include a processed cartilageselected from the group consisting of a ground cartilage, a mincedcartilage, a cartilage paste and combinations thereof. The processedcartilage may be an autograft cartilage, an allograft cartilage orcombinations thereof. When processed cartilage is added to a minced orground placental membrane preparation, the processed cartilage ispreferably provided in between a 3:1 and a 1:3 ratio by volume to theoriginal membrane preparation. In an additional embodiment, the minced,ground or morselized membrane particles may have an average particlesize of less than about 1.5 mm and are absorbed to a collagen matrix.The collagen matrix may them be used in a method of treating wounds, aswill be described in more detail below.

The placental membrane preparation may include hyaluronic acid, salineor a combination thereof. Hyaluronic acid and saline may be includedwith the placental membrane preparation when it is desired to inject thepreparation into a skeletal joint. When hyaluronic acid or saline isadded to a placental membrane preparation, the hyaluronic acid or salineis preferably provided in a 2:1 or 1:1 ratio by volume to the originalmembrane preparation.

The placental membrane preparation may include one or more biocompatibleglues. Biocompatible glues are natural polymeric materials that act asadhesives. Biocompatible glues may be formed synthetically frombiological monomers such as sugars and may consist of a variety ofsubstances, such as proteins and carbohydrates. Proteins such as gelatinand carbohydrates such as starch have been used as general-purpose gluesfor many years. Preferably, the biocompatible glue is fibrin glue, suchas Tisseel. Fibrin is made up of fibrinogen (lyophilized pooled humanconcentrate) and may also include thrombin (which may be reconstitutedwith calcium chloride).

C. THE HYPOTHERMIC STORAGE SOLUTION

The membrane may be stored in a hypothermic storage solution allowingthe preservation of the viability of the membrane cells. The solutionpresently described is effective in storing tissues at temperaturesabove freezing, for instance at a temperature ranging from 0° C. to 20°C. In an additional embodiment, the solution is effective attemperatures ranging between more than 0° C. and less than 10° C. In oneexemplary embodiment, the hypothermic storage solution may include acommercially available tissue culture media with appropriatesupplements. All components of the solution are chemically defined/cGMPcompliant and free of both xeno-derived and human-derived components. Inthis embodiment, the tissue culture media comprises DMEM. DMEM containsamino acids, salts (calcium chloride, potassium chloride, magnesiumsulfate, sodium chloride, and monosodium phosphate) and highconcentrations of glucose and vitamins (folic acid, nicotinamide,riboflavin, B12). Additionally, it contains iron and phenol red. DMEM issuitable for most types of cells, including human, monkey, hamster, rat,mouse, chicken, and fish cells. DMEM is commercially available fromseveral providers, for instance Corning, Inc. (Tewksbury Mass., Catalog#15-018).

The hypothermic storage solution of the present invention furtherincludes supplementation with appropriate buffers to maintainphysiological pH despite changes in carbon dioxide concentrationproduced by cellular respiration. In one embodiment, the buffercomprises HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid), a zwitterionic organic chemical buffering agent. HEPES is widelyused in cell culture, largely because it is better at maintainingphysiological pH as compared to bicarbonate buffers. The HEPES buffermay be used at a concentration as will be known to one of skill in theart, for example from about 20 mM to 30 mM. In one embodiment, thesolution contains a concentration of 25 mM HEPES.

The hypothermic storage solution further includes supplementation withalbumin. In one embodiment, the albumin is human plasma albumin. In anadditional embodiment, the human plasma albumin is recombinant humanalbumin (rhA), for instance Cellastim rhA manufactured by InVitria(Junction City, Kans.). The human plasma albumin may be used at aconcentration from about 2.0 g/L to about 3.0 g/L. In one embodiment,the solution contains a concentration of about 2.5 g/L rhA. In anotherembodiment, the solution contains a concentration of at least 3.0 g/LrhA.

D. STORAGE OF THE PLACENTAL MEMBRANE

In one embodiment, the invention is directed to a method of storing aprepared placental membrane, the method including placing the membranein a sterile container including a hypothermic storage solution. In thisembodiment, the storage solution contains a commercially available cellculture medium, such as DMEM, HEPES buffer at a concentration between 20mM to 30 mM and rhA at a concentration between about 2.0 g/L to about3.0 g/L. In an additional embodiment, the sterile container is sealedwith a volume of air in contact with the storage solutions such that theratio of storage solution and air is between about 2:1 and 5:1 (solutionvolume: air volume).

In this method, the placental membrane preparation may be stored andtransported in the hypothermic storage solution of the presentinvention. In one embodiment, the membrane and storage solution areplaced in a sterile container, such as a jar or tray, and sealed toprevent contamination. A volume of air is included within the sealedcontainer to allow for continued metabolism by the cells within themembrane. The ratio of the volume of storage solution to air may vary,as will be known to one of skill in the art. In one embodiment, thisratio may be between about 2:1 and 5:1 (solution volume: air volume). Inanother embodiment, the ratio is about 3:1. The container may bepositioned and sealed within a second container to provide greaterprotection from contaminants. For example, the membrane may behypothermically stored between two sterile nested trays (the“tray-in-tray” configuration) or stored between a sterile jar and asterile tray (the “jar-in-tray” configuration).

The human albumin contained within the presently described storagesolution maintains the viability of the placental membrane cells for anextended period of time. In this embodiment, the placental membranestored within the hypothermic storage solution exhibits a ratio of liveto dead cells after an extended period of storage, as illustrated inTable 1 below.

TABLE 1 Ratio of live to dead cells after extended storage times. Ratioof Live Cells to Dead Cells Hours of Hypothermic Storage >1.5 36 >1.0120 >2.0 360 >1.5 720 >1.0 1,000In an additional embodiment, the placental membrane exhibits cellviability in the range of 45% to 85% following one thousand hours ofbeing hypothermically stored in the solution.

In one embodiment, the storage solution of the method significantlyincreases membrane integrity, reduces osmotic swelling, and retains cellviability over time as compared to solutions that do not contain humanalbumin. For example, the inclusion of human albumin in the solutionpreserves the thickness, reduces swelling, reduces damage to theepithelial cell layer and promotes cell retention within theextracellular matrix of the placental membrane. Increasing membraneintegrity and preserving cell viability may be accomplished by theaddition of an effective amount of human plasma albumin to thehypothermic storage solution. In addition, the human plasma albumin actsto preserve the total protein content of the placental membrane. In oneembodiment, the hypothermic storage solution herein described maintainsa total protein content of more than about 450 ng of protein per mg ofplacental membrane following twenty-four hours of being hypothermicallystored in the solution. In an additional embodiment, the storagesolution maintains a total protein content of more than about 300 ng ofprotein per mg of placental membrane following one thousand hours ofbeing hypothermically stored in the solution (see FIG. 5E).

In an additional embodiment of the method, the addition of the humanplasma albumin acts to maintain activity of tissue inhibiting matrixmetalloproteinases (TIMPs) 1, 2 and 4. TIMPs are known inhibitors ofmatrix metalloproteinases (MMPs), which are responsible forenzymatically breaking down various ECM proteins. Maintenance of TIMPactivity, and reduction of extracellular matrix proteins, could act topreserve membrane activity over time. In this embodiment, the placentalmembrane preserved in the storage solution described herein has a totalTIMP 1, 2 and 4 concentration of more than 400 ng per mg of placentalmembrane following twenty-four hours of storage in the hypothermicstorage solution of the invention. In an additional embodiment, theplacental membrane preserved in the storage solution described hereinhas a total TIMP 1, 2 and 4 concentration of more than 250 ng per mg ofplacental membrane following one thousand hours of storage in thehypothermic storage solution of the invention (see FIG. 5F, describedherein).

In an additional embodiment, the placental membrane of the method may beminced, ground or morselized prior to storage in the hypothermic storagesolution, as described herein, allowing the preservation of theviability of some membrane cells. Additionally, the placental membranepreparation may further include a processed cartilage selected from thegroup consisting of a ground cartilage, a minced cartilage, a cartilagepaste and combinations thereof. The processed cartilage may be anautograft cartilage, an allograft cartilage or combinations thereof.When processed cartilage is added to a minced or ground placentalmembrane preparation, the processed cartilage is preferably provided inbetween a 3:1 and a 1:3 ratio by volume to the original membranepreparation. In an additional embodiment, the minced, ground ormorselized membrane may be arranged in particles with a size of lessthan about 1.5 mm and absorbed to the collagen matrix before storage inthe hypothermic storage solution. The collagen matrix may them be usedin a method of treating wounds, as will be described in more detailbelow. In addition, the placental membrane tissue is at least partiallysubmerged in the storage solution including DMEM and at least about0.025% w/v human plasma albumin.

In an additional embodiment, the invention includes a system comprisinga placental membrane hypothermically stored within a container. Thecontainer includes a hypothermic storage solution including a commercialavailable tissue culture medium supplemented with human plasma albuminand an appropriate buffer to maintain physiological pH. In oneembodiment, the human plasma albumin is recombinant human albumin at aconcentration from about 2.0 g/L to about 3.0 g/L. In one embodiment,the solution contains a concentration of about 2.5 g/L rhA. In anotherembodiment, the solution contains a concentration of at least 3.0 g/LrhA. In this embodiment, the buffer comprises HEPES buffer at aconcentration from about 20 mM to 30 mM. In one embodiment, the solutioncontains HEPES at a concentration of 25 mM.

The sterile container for holding the placental membrane and thesolution may be sealed to prevent contamination. A volume of air isincluded within the sealed container to allow for continued metabolismby the cells within the membrane. The ratio of the volume of storagesolution to air may vary, as will be known to one of skill in the art.In one embodiment, this ratio may be between about 2:1 and 5:1 (solutionvolume: air volume). In another embodiment, the ratio is about 3:1. Thecontainer may be positioned and sealed within a second container toprovide greater protection from contaminants. The system may furtherinclude HEPES buffer as a component of the storage solution, where theHEPES buffer is at a concentration between 20 mM and 30 mM.

The human albumin contained within the presently described storagesolution of the system maintains the viability of the placental membranecells for an extended period of time. In this embodiment, the placentalmembrane stored within the hypothermic storage solution exhibits a ratioof live to dead cells after an extended period of storage, asillustrated in Table 1. In an additional embodiment, the inclusion ofhuman albumin in the solution preserves the thickness, reduces swelling,reduces damage to the epithelial cell layer and promotes cell retentionwithin the extracellular matrix of the placental membrane bysignificantly increasing membrane integrity, reducing osmotic swelling,and retaining cell viability over time as compared to solutions which donot contain human albumin. The human plasma albumin acts to preserve thetotal protein content of the placental membrane. In one embodiment, thehypothermic storage solution of the system maintains a total proteincontent of more than about 450 ng of protein per mg of placentalmembrane following twenty-four hours of being hypothermically stored inthe solution. In an additional embodiment, the storage solution includedin the system maintains a total protein content of more than about 300ng of protein per mg of placental membrane following one thousand hoursof being hypothermically stored in the solution (see FIG. 5E).

In another embodiment, the addition of the human plasma albumin to thestorage solution of the system acts to maintain activity of TIMPS 1, 2and 4. In this embodiment, the placental membrane preserved in thestorage solution of the system has a total TIMP 1, 2 and 4 concentrationof more than 400 ng per mg of placental membrane following twenty-fourhours of storage. In an additional embodiment, the placental membranepreserved in the storage solution of the system has a total TIMP 1, 2and 4 concentration of more than 250 ng per mg of placental membranefollowing one thousand hours of storage (see FIG. 5F, described herein).

The placental membrane preparation included in the system may include aprocessed cartilage selected from the group consisting of a groundcartilage, a minced cartilage, a cartilage paste and combinationsthereof, as described in detail above. In an additional embodiment, theminced, ground or morselized membrane particles may have an averageparticle size of less than about 1.5 mm and may then be absorbed to acollagen matrix. In addition, the placental membrane of the system maybe meshed so that it may be stretched to increase its length and width.The membrane of the system may comprise both the amniotic and chorioniclayers. The spongy layer of the amnion tissue may be removed or mayremain intact depending on the intended application for the preservedtissue. In another example, the chorion of the placental membrane hasbeen removed. In another example, the epithelial layer has been removedfrom the placental membrane.

E. TREATMENT OF WOUNDS AND DEFECTS

The embodiments of the placental membrane preparation, described herein,may be used to regenerate damaged tissues. The storage solution andmethods may be utilized with placental membranes used in a number ofclinical conditions including, but not limited to, traumatic injury,such as lacerations or burns.

The placental membrane in the hypothermic storage solution may act as ascaffold or matrix for cell engraftment and in-growth. Thus, thepreserved membranes may act as an integral matrix with cells intact intheir normal location and without culturing. The placental membranesalso provide a reservoir of growth factors attracting incomingblood-born MSCs, chondrocytes, and other reparative cells.

In one embodiment, the present invention includes a method for treatinga wound comprising the application of the placental membrane tissuestored as described above to a wound. In certain embodiments, theplacental membrane comprises an amniotic membrane. Here, the stromalsurface of the tissue is applied to the wound to promote healing.Alternatively, the membrane tissue may comprise cartilage.

In an additional embodiment, the placental membrane may be ground,minced or morselized using techniques known in the art, prior tohypothermically storing the placental membrane in the storage solution.Such membranes may be further combined with ground or minced autograftor allograft cartilage for implantation into the wound or defect.

In an additional embodiment, the placental membrane may be stored withinthe hypothermic solution for an extended period of time without freezingthe membrane. In this embodiment, the membrane is not frozen prior to orafter being hypothermically stored in the solution.

In a further embodiment, the present invention is directed to a systemfor treating a wound comprising a placental membrane hypothermicallystored within a container. The system includes a storage solution ofDMEM and human plasma albumin. In an additional embodiment the humanplasma albumin is recombinant human albumin. The placental membrane ofthe system exhibits ratios of live to dead cells after extended storagetimes as illustrated in Table 1. In an additional embodiment, theaddition of the human plasma albumin to the solution within the systemacts to maintain activity of TIMPs 1, 2 and 4. TIMPs are knowninhibitors of MMPs, which are responsible for enzymatically breakingdown various ECM proteins. Maintenance of TIMP activity, and reductionof extracellular matrix proteins, could act to preserve membraneactivity over time.

In an additional embodiment, the placental membrane within the systemmay be ground, minced or morselized using techniques known in the art,prior to hypothermically storing the placental membrane in the storagesolution. Such membranes may be further combined with ground or mincedautograft or allograft cartilage for implantation into a defect.

EXPERIMENTAL A. Definitions

“Cell Viability” is defined as the percentage of surviving cells out oftotal cells in a cell suspension. Defined as

${V = {\frac{L}{\left( {L + D} \right)}*100\%}},$

where V is the viability percentage, L is the number live cells, and Dis the number of dead cells in a suspension.

“H&E stain” is defined as hematoxylin and eosin stain.

“Modulus” is defined as the extent to which an object resistsdeformation in response to an applied force. Defined as

$M = {\frac{\sigma}{e}.}$

“Strain” is defined as the normalized measure of deformation defined asthe change in length divided by the original length; e=ΔL/L.

“Stress” is

${\sigma = \frac{F}{A}},$

defined as where F is the force (N) acting on the area (A, cm²).

“Total Protein Content” is defined as the total level of 40 knowncytokines. To remove bias, protein levels were normalized by tissueweight. Levels are defined as

$T = {\sum\limits_{{Protein}\mspace{11mu} 1}^{{Protein}\mspace{14mu} 40}\; \frac{P_{n}}{W}}$

where T is the total protein content, P is the concentration of eachprotein, and W is the weight of the tissue sample.

B. Preliminary Cell Viability Study

Preliminary studies evaluating storage of amniotic membrane inhypothermic conditions were performed using the following groups:

(1) Cellgenix CellGro

(2) Hypothermosol

(3) RPMI

(4) DMEM (containing 25 mM HEPES buffer)

(5) fully-supplemented DMEM (containing 25 mM HEPES Buffer, Insulin,Transferrin, Selenium, 2 mM L-glutamine, and 0.25% w/v rhA)

(6) DMEM (containing 25 mM HEPES buffer and 0.25% w/v rhA).

In this preliminary study, a Multitox Fluor Viability kit was used todetermine live cell viability over time to serve as an indication ofmembrane integrity. FIG. 1A illustrates the results, graphed as a ratioof Multitox live cells to total DNA count (as determined by a PicogreendsDNA assay). The DMEM+recombinant human albumin (rhA) solution was mosteffective at retaining cell viability over time. FIGS. 1B-1D illustratequalitative imaging of the three conditions with the best viability attheir endpoints. As illustrated in these images, the DMEM+rhA solutionhas considerable viability at 42 days. These results show that thehypothermic storage solution containing DMEM+rhA demonstrates superiorviability as compared to the other storage media.

C. Cell Viability as a Measure of Membrane Integrity

1. Multitox Assay

In this experiment cell viability was used as a measure for membraneintegrity.

Preliminary data suggested that DMEM+rhA would be the best combinationfor preserving membrane integrity. DMEM alone and DMEM+rhA containedwithin (a) a “jar-in-tray” or (b) “tray-in-tray” arrangement wereinvestigated. Similar oxygen to storage media ratios were maintained forboth groups. Therefore, the main variable between storage conditions wasthe volume of storage media used. The “jar-in-tray” configurationallowed for a storage solution volume of 12 mL of media in a 15 cc jar,while the “tray-in-tray” configuration allowed for a significantlyhigher volume of storage media 30 mL of media in a 40 cc tray.

At days 1 and 42 membranes stored in the different storage media andpackaging configurations were subjected to a PBS rinse followed bydigestion in collagenase to free the cells from the membrane. The cellswere then directly assessed for viability using the Multitox viabilityassay. FIG. 2A illustrates the viability of cells isolated fromplacental membranes harvested from four donors. The solid black line atapproximately 55% viability indicates the average viability at day 1,while the broken lines and shaded area indicate the standard deviation.Several trends were evident in evaluating this data. The datademonstrates that DMEM+rhA retained higher cell viability than DMEMalone across both storage conditions and the “Tray-in-Tray”configuration trended towards higher viability as compared to the“Jar-in-Tray” configuration. The membrane stored in DMEM+rhA solution inthe “Tray-in-Tray” configuration retained the highest amount ofviability at 42 days. The “Tray-in-Tray” and “Jar-in-Tray” samples werecombined to evaluate whether there was a significant difference betweenthe storage conditions. FIG. 2B illustrates that the DMEM+rhA solutionhad significantly higher viability than DMEM alone. There was asignificant difference between the two packaging conditions with the“Tray-in-Tray” configuration resulting in significantly higher viabilitythan the Jar in Tray configuration, likely due to a higher volume ofstorage media (FIG. 2C).

2. Cell Imaging

As a secondary measure to verify cell viability post membrane digestionat 42 days of storage, cells remaining after the Multitox viabilitytesting were seeded onto a 24-well plate and cultured (37° C., 5% CO₂)in standard growth media (DMEM with 1% Penicillin, 1% Streptomycin, and1% Amphotericin, 2 mM L-glutamine, and 20% FBS). After 48-72 hours,cells were stained using Calcein AM and imaged under a fluorescentmicroscope to qualitatively analyze cell viability. The trends from themultitox viability studies held true for qualitative evaluation of celldensity after 42 days of hypothermic storage. Specifically, it wasdemonstrated that at Day 42, the “Tray-in-Tray” packaging configurationexhibited improved viability over the “Jar-in-Tray” packagingconfiguration and the DMEM+rhA solution exhibited improved cellviability over DMEM alone (FIG. 3).

D. H&E Staining and Microscopic Inspection of Membrane Integrity

In addition to quantitative comparisons of membrane integrity,macroscopic handling characteristics were assessed and H&E staining wasperformed to evaluate preservation of membrane integrity after extendedperiods of hypothermic storage. Some qualitative handlingcharacteristics of amniotic membrane samples stored for 42 days were:(1) samples in DMEM+rhA were consistently thicker and more like tissueat 1 day; and (2) amniotic membrane samples stored in DMEM alone seemedthinner.

To more closely examine membrane integrity, amniotic membrane stored inDMEM or DMEM+rhA for 4 or 6 weeks were cross sectioned and stained withhematoxylin and eosin (H&E). Of note, 4 and 6 week staining of amnioticmembrane was done at different times; therefore, the staining intensityvaries between 4 and 6 week samples. There were clear differences in theamniotic membranes stored in DMEM (FIGS. 5A and 5C) and those stored inDMEM+rhA (FIGS. 5B and 5D) at both 4 and 6 week time points. Thesedifferences included: (1) more damage to epithelial layer of membranesstored in the DMEM solution alone; (2) the thickness of the amnioticmembrane was preserved more efficiently in membranes stored in theDMEM+rhA solution; and (3) more cells were retained within the ECM ofamniotic membranes stored in the DMEM+rhA solution. Overall, tissuesstored in the DMEM+rhA solution appeared larger and thicker as comparedto tissues stored in DMEM only.

E. Proteomics Assay to Evaluate Protein Content

Total protein content of the stored placental membrane cells was nextassessed to determine whether storage conditions affected the growthfactor content of the membranes over time. Membrane tissues were weighedat predetermined time points. The tissues were then cryo-homogenized andtotal protein content was extracted using a total protein extractionbuffer. Total protein was then measured using a custom quantibodymicroarray to quantitatively assess 40 separate cytokines simultaneouslyfor each sample. For each sample, the levels of all 40 cytokines weresummed and normalized by the sample weight to determine the total amountof protein in picograms per milligram of membrane (FIG. 5E), with eachcondition assessed in duplicate.

According to the proteomics data means, trends similar to those seenwith histology, cell viability, and cell viability imaging wereobserved. The concentrations of tissue inhibiting TIMPs 1, 2, and 4 weresummed for each sample and averaged for each storage solution condition(FIG. 5F). TIMPs are known inhibitors of MMPs, which are responsible forenzymatically breaking down various ECM proteins. Overall, it appearsthat maintenance of TIMP activity could be a potential mechanism for thepreservation of membrane integrity over time.

F. Mechanical Testing to Evaluate Membrane Integrity

Mechanical testing was completed on samples stored in the DMEM storagesolution at Day 1. These values were compared to data collected formembranes stored in DMEM and DMEM+rhA at 9 weeks. Briefly, an MTS wasused to report the time, displacement, and force every 1/100th of asecond. The maximum values for force and the maximum displacement werecalculated at the time of membrane failure. Using the maximumdisplacement and the initial distance between the clamps, strain wascalculated: e=(Lf−Li)/Li (FIG. 6B). Stress is defined as and wascalculated using maximums from the data collected during tensile testing(FIG. 6A). Finally, the modulus of elasticity (‘Modulus’=stress/strain)is the mathematical description of an object's tendency to be deformedelastically when a force is applied to it (FIG. 6C).

Stress, strain and modulus were calculated and evaluated for all datapoints. Interestingly, for all mechanical properties there weresignificant differences between the amniotic membrane samples stored inthe DMEM solution and those stored in DMEM+rhA solution. Additionally,amniotic membrane samples hypothermically stored in the DMEM+rhAsolution were not significantly different in their mechanical propertiescompared to mechanical properties of amniotic membrane at day 1. Thecells stored in the DMEM+rhA solution were able to sustain the largeststress, underwent less strain, and had a greater modulus than thosestored in the DMEM solution. Importantly, these characteristics matchedwhat was seen in tissue stored for only 1 day. These results imply thatsamples stored in DMEM+rhA had less tissue degradation than those storedin DMEM alone. These data suggest that the DMEM+rhA storage solutionresults in less destruction and/or better preservation of the membranethan DMEM alone.

Conclusion

All tests performed indicate that the integrity of the amniotic membranewhen subjected to hypothermic storage conditions is best preserved whenusing the DMEM+rhA solution compared to DMEM solution. This conclusionis supported by data from cell viability studies, H&E staining,proteomics microarrays, and mechanical testing.

G. Animal Studies EXPERIMENTAL

Placental membrane samples were examined for their effectiveness inpromoting wound healing. Four 15 mm diameter, full thickness wounds werecreated on the dorsal skin surface (i.e. the back) of three laboratoryrats. Four types of placental membrane preparations were placed stromalside down on the wounds of each animal—one type of membrane on eachwound. The following types of membranes were utilized:

(1) Fresh placental membrane stored in a hypothermic solution containingDMEM, 25 mM HEPES and 2.5 g/L rhA after 25 days storage;

(2) Dehydrated meshed placental cell preparation;

(3) Dehydrated fully intact amnion; and

(4) Control (no membrane)

A primary dressing (cuticerin) was secured to the skin using staples tocover the wounds.

The wounds were inspected nine and twenty-one days post-procedure andhistological samples of the wounds were collected and evaluated forwound healing.

Results

Nine days post-procedure the wounds treated with the fresh storedmembrane, dehydrated mesh and dehydrated amnion appeared smaller thanthose of the control group (FIGS. 7B-7D). FIG. 7A provides a guideindicating placement of the membranes on the wounds. Referring now toFIG. 8, histological examination indicates that the wounds treated withthe fresh stored membrane and the dehydrated mesh samples showed morecellular granulation tissue and re-epithelialization as compared to thecontrol. Specifically, referring to FIG. 13A, the wounds treated withfresh stored membrane exhibited substantially less abnormal tissuecompared to wounds treated with dehydrated mesh, dehydrated amnion, andcontrol. Conversely, referring to FIG. 13B, the wounds treated withfresh stored membrane exhibited greater basket-weave matrix than theother three treatment groups, although the number of follicles andsebaceous glands in wounds treated with fresh stored membrane were notsignificantly different than the other treatment groups at nine days(FIG. 13C).

Twenty-one days post-procedure the wounds treated with the fresh storedmembrane were smaller as compared to the wounds of the dehydrated mesh,dehydrated amnion, and control groups (FIGS. 9A-9D). The results of the21-day post-procedure histological examination are illustrated in FIGS.10-12. As shown in FIG. 10, the wound treated with fresh storedplacental membrane showed increased wound healing as compared to theother samples. Referring to FIG. 11C, the histological testing of theuntreated wound illustrated granulation tissue and a thin area ofepithelialization. The wounds treated with the dehydrated mesh (FIG.11D) and the dehydrated amnion (FIG. 11A) preparations showed onlyslightly increased granulation and epithelialization as compared to thecontrol. In contrast, the wound treated with the fresh stored placentalmembrane (FIG. 11B) illustrated increased collagen deposition and theextensive development of hair follicles and sebaceous glands. Thesechanges are an indication of scarless regenerative wound healing. FIG.12D illustrates this increased development of hair follicles andsebaceous glands as well as new collagen deposition and the formation ofgranulation tissue in the wound treated with the fresh stored placentalmembrane. In contrast, these developments are not present in the woundstreated with the control (FIG. 12A), the dehydrated mesh (FIG. 12B) orthe dehydrated amnion (FIG. 12C) preparations. Further, referring toFIG. 13A, although all treatment groups exhibited less abnormal tissueat twenty-one days compared with nine days, the wounds treated withfresh stored membrane continued to exhibit less abnormal tissue comparedto wounds treated with dehydrated mesh, dehydrated amnion, and control.Similarly, referring to FIG. 13B, although the wounds of all treatmentgroups exhibited greater basket-weave matrix, the wounds treated withfresh stored membrane exhibited greater basket-weave matrix than theother three treatment groups. Unlike at nine days, at twenty-one days,the wounds treated with fresh stored membrane exhibited a greater numberof follicles and sebaceous glands than in wounds treated with dehydratedmesh and control (FIG. 13C).

Conclusion

It is clear from the representative histological images (FIGS. 10-12)and from the quantification thereof (FIGS. 13A-13C) that treatment ofthe wound with the fresh stored placental membrane scaffold resulted inthe best skin regeneration response as compared to the dehydrated amnionand dehydrated mesh preparations. The wounds treated with the freshstored membrane showed evidence of re-epithelialization at nine days.The epithelial layer in these wounds developed into a thick stratifiedepidermis with finger-like projections that extended into the dermallayer, representative of a healthy epidermal healing. In contrast, theother preparations showed only limited re-epithelialization across thewound bed at nine days post-procedure.

Blood vessel presence appeared to be greater at the nine day time pointas compared to the twenty-one day time point for all groups. Thedehydrated amnion appeared to provide the highest level of vesselformation. It is apparent that the tissue was being remodeled and thevessels were breaking down as the wound healing process progressed.

These data suggest that the fresh, hypothermically stored placentalmembranes expedited the wound healing process. Further, the fresh storedgrafts enhanced the body's ability to regenerate skin tissue thatclosely mimics unwounded skin. This is apparent by the early epidermal,hair follicle and gland formation, as well as the high degree of basketweave matrix. The fresh hypothermically stored placental preparationsillustrated a strikingly greater response in wound healing.

What is claimed is:
 1. A tissue storage method comprisinghypothermically storing a placental membrane in a solution includingDulbecco's modified Eagle's medium (DMEM) and human plasma albumin. 2.The method according to claim 1, wherein the human plasma albumin isrecombinant human albumin (rhA).
 3. The method according to claim 1,wherein the placental membrane is hypothermically stored in the solutionat a temperate ranging between more than 0° C. and less than about 10°C.
 4. The method according to claim 1, wherein the placental membraneand the solution are hypothermically stored in a sealed container. 5.The method according to claim 1, comprising storing a placental membranein a solution including DMEM and human plasma albumin for at least 14days.
 6. The method according to claim 1, wherein the placental membraneexhibits a ratio of live cells to dead cells of greater than 1.5following thirty-six hours of being hypothermically stored in thesolution.
 7. The method according to claim 1, wherein the placentalmembrane exhibits a ratio of live cells to dead cells greater than 1.0following one hundred twenty hours of being hypothermically stored inthe solution.
 8. The method according to claim 1, wherein the placentalmembrane exhibits a ratio of live cells to dead cells greater than 2.0following three hundred sixty hours of being hypothermically stored inthe solution.
 9. The method according to claim 1, wherein the placentalmembrane exhibits a ratio of live cells to dead cells of greater thanabout 1.5 following seven hundred twenty hours of being hypothermicallystored in the solution.
 10. The method according to claim 1, wherein theplacental membrane exhibits a ratio of live cells to dead cells of about1.0 or more following one thousand hours of being hypothermically storedin the solution.
 11. The method according to 1, claim wherein theplacental membrane exhibits a cell viability in a range between about45% and 85% following one thousand hours of being hypothermically storedin the solution.
 12. The method according claim 1, further comprisingpromoting preservation of membrane integrity of the placental membraneby including an effective amount of the human plasma albumin in thesolution.
 13. The method according to claim 12, wherein promotingpreservation of membrane integrity of the placental membrane includespromoting preservation of a thickness of the placental membrane,reducing damage to an epithelial cell layer of the placental membraneand promoting cell retention within an extracellular matrix of theplacental membrane.
 14. The method according to claim 1, furthercomprising promoting cell viability of the placental membrane byincluding an effective amount of the human plasma albumin in thesolution.
 15. The method according to claim 1, further comprisingpromoting preservation of total protein content of the placentalmembrane by including an effective amount of the human plasma albumin inthe solution.
 16. The method according to claim 1, wherein the placentalmembrane has a total protein content of more than about 450 ng ofprotein per mg of the placental membrane following twenty-four hours ofbeing hypothermically stored in the solution.
 17. The method accordingto claim 16, wherein the placental membrane has a total protein contentof more than about 300 ng of protein per mg of the placental membranefollowing one thousand hours of being hypothermically stored in thesolution.
 18. The method according to claim 1, further comprisingpromoting preservation of extracellular matrix proteins of the placentalmembrane by including an effective amount of the human plasma albumin inthe solution.
 19. The method according to claim 1, further comprisingpromoting a reduction of extracellular matrix metalloproteinases withinthe placental membrane by including an effective amount of the humanplasma albumin in the solution.
 20. The method according to claim 1,further comprising promoting preservation of tissue inhibitor of matrixmetalloproteinases 1, 2 and 4 within the placental membrane by includingan effective amount of the human plasma albumin in the solution.
 21. Themethod according to claim 1, wherein the placental membrane has a tissueinhibitor of matrix metalloproteinases 1, 2 and 4 protein content ofmore than 400 ng of protein per mg of the placental membrane followingtwenty-four hours of being hypothermically stored in the solution. 22.The method according to claim 1, wherein the placental membrane has atissue inhibitor of matrix metalloproteinases 1, 2 and 4 protein contentof more than 250 ng of protein per mg of the placental membranefollowing one-thousand hours of being hypothermically stored in thesolution.
 23. The method according to claim 1, wherein the placentalmembrane is hypothermically stored between nested trays.
 24. The methodaccording to claim 1, comprising morselizing the placental membraneprior to hypothermically storing the placental membrane in the solution.25. The method according to claim 24, comprising adsorbing themorselized placental membrane to a mammalian collagen matrix prior tohypothermically storing the placental membrane in the solution.
 26. Themethod according to claim 1, comprising adding minced cartilage to thesolution.
 27. The method according to claim 1, wherein the placentalmembrane is not frozen prior to, during or after being hypothermicallystored in the solution.
 28. A method for treating wounds, comprising theapplication of tissue stored by the method according to claim 1 andapplying the tissue to the wound.
 29. The method according to claim 28,wherein the tissue is amniotic membrane.
 30. The method according toclaim 28, wherein the tissue is cartilage.
 31. The method of accordingto claim 28, wherein the stromal surface of the tissue is applied to thewound.
 32. A tissue storage system comprising a placental membranehypothermically stored within a container including a solution of DMEMand human plasma albumin.
 33. The system according to claim 32, whereinthe placental membrane exhibits a ratio of live cells to dead cellsgreater than 1.5 following thirty-six hours of being hypothermicallystored in the solution, a ratio of live cells to dead cells greater than1.0 following one hundred twenty hours of being hypothermically storedin the solution, a ratio of live cells to dead cells greater than 2.0following three hundred sixty hours of being hypothermically stored inthe solution, a ratio of live cells to dead cells greater than about 1.5following seven hundred twenty hours of being hypothermically stored inthe solution and a ratio of live cells to dead cells of about 1.0 ormore following one thousand hours of being hypothermically stored in thesolution.
 34. The system according to claim 32, wherein the placentalmembrane has a total protein content of more than about 450 ng ofprotein per mg of the placental membrane following twenty-four hours ofbeing hypothermically stored in the solution and a total protein contentof more than about 300 ng of protein per mg of the placental membranefollowing one thousand hours of being hypothermically stored in thesolution.
 35. The system according to claim 32, wherein the placentalmembrane has a tissue inhibiting matrix metalloproteinases 1, 2 and 4protein content of more than 400 ng of protein per mg of the placentalmembrane following twenty-four hours of being hypothermically stored inthe solution and a tissue inhibiting matrix metalloproteinases 1, 2 and4 protein content of more than 250 ng of protein per mg of the placentalmembrane following one thousand hours of being hypothermically stored inthe solution.
 36. The system according to claim 35, wherein thecontainer includes a pair of sealed trays within which the placentalmembrane is situated.
 37. The system according to claim 35, wherein theplacental membrane is arranged into pieces having an average particlesize of less than about 1.5 mm that are adsorbed to a porous collagenmatrix.
 38. The system according to claim 37, wherein the placentalmembrane is a meshed placental membrane.
 39. The system according toclaim 32, wherein the placental membrane includes an amnion layer and achorion layer.
 40. The system according to claim 32, wherein theplacental membrane includes a spongy layer.
 41. The system according toclaim 32, wherein the solution contains HEPES buffer.
 42. A tissuecomprising: a placental membrane exhibiting a ratio of live cells todead cells greater than about 1.5 following two hundred forty hours ofbeing hypothermically stored in a solution.
 43. The tissue according toclaim 42 comprising a ratio of live cells to dead cells greater thanabout 1.5 following seven hundred twenty hours of being hypothermicallystored in the solution.
 44. The tissue according to claim 42, whereinthe tissue is at least partially submerged in a storage solutionincluding DMEM and at least about 0.025% w/v human plasma albumin.